Combination pharmaceutical composition and methods of treating diseases or conditions associated with neurodegenerative diseases

ABSTRACT

The present invention relates to combination pharmaceutical composition comprising an activated-potentiated from of an antibody to gamma interferon, and an activated-potentiated form of an antibody to S-100 protein and method of treating multiple sclerosis and other neurodegenerative diseases, as well as the diseases and conditions associated with neuroinfections.

FIELD

The present invention relates to a combination pharmaceuticalcompositions and method of treating multiple sclerosis and otherneurodegenerative diseases, as well as the diseases and conditionsassociated with neuroinfections.

BACKGROUND

Multiple sclerosis or MS is a chronic disease that affects the brain andspinal cord resulting in loss of muscle control, balance, and sensation(such as numbness). Currently, the exact cause of MS remains unknown,but researchers believe that a combination of several factors may beinvolved. It is believed that MS appears in genetically predisposedindividuals possibly in the presence of specific external factors whichlead to the development of MS. It is now generally accepted that MSinvolves an autoimmune process—an abnormal response of the body's immunesystem that is directed against the myelin (the fatty sheath thatsurrounds and insulates the nerve fibers) in the central nervous system(CNS—the brain, spinal cord and optic nerves). MS results in a thinningor complete loss of myelin and, as the disease advances, the cutting(transection) of the neuron's extensions or axons. When the myelin islost, a neuron can no longer effectively conduct electrical signals. Arepair process, called remyelination, takes place in early phases of thedisease, but the cell's myelin sheath cannot be completely rebuilt.Repeated attacks lead to successively fewer effective remyelinations,until a scar-like plaque is built up around the damaged axons.

Apart from demyelination, the other pathologic trait of the disease isinflammation. According to a strictly immunological explanation, theinflammatory process is caused by T cells, a kind of lymphocyte.Lymphocytes are cells that play an important role in the body'sdefenses. In MS, T cells gain entry into the brain via the blood-brainbarrier. It is believed that the T cells recognize myelin as foreign andattack it which triggers inflammatory processes, stimulating otherimmune cells and soluble factors like cytokines and antibodies.

In addition to autoimmune disorder, some researchers believe thatinfections may somehow trigger the immune system to attack nerve cells.Basically, it is believed that the virus (or a bacterium) that causes aninitial infection “looks” like a nerve cell. The immune system developsT-cells to fight off the virus. Those T-cells remain in the body afterthe infection is gone and become confused when they “see” a nerve cell,mistaking it for an invader. The result is that your immune systemattacks the nervous system.

There are four main varieties of MS. 1. Relapsing/Remitting (RRMS):characterised by relapses during which time new symptoms can appear andold ones resurface or worsen. 2. Secondary Progressive (SPMS):characterized by a gradual worsening of the disease between relapses. 3.Progressive Relapsing Multiple Sclerosis (PRMS): This form of MS followsa progressive course from onset, punctuated by relapses. 4. PrimaryProgressive (PPMS): This type of MS is characterized by a gradualprogression of the disease from its onset with no remissions at all.

There is no known cure for MS at this time. However, there are therapiesthat may slow the disease. The goal of treatment is to control symptoms.Medications that alter the immune system, for example interferons, havebeen used to manage multiple sclerosis. Interferons are proteinmessengers that cells of the immune system manufacture and use tocommunicate with one another. There are different types of interferons,such as alpha, beta, and gamma. All interferons have the ability toregulate the immune system and play an important role in protectingagainst intruders including viruses. Each interferon functionsdifferently, but the functions overlap. The beta interferons have beenfound useful in managing multiple sclerosis.

There is a continuing need for new drug products with desiredtherapeutic efficacy for treatment of MS and related symptoms.

The therapeutic effect of an extremely diluted form (or ultra-low form)of antibodies potentized by homeopathic technology (activatedpotentiated form) has been discovered by the inventor of the presentpatent application, Dr. Oleg I. Epshtein. U.S. Pat. No. 7,582,294discloses a medicament for treating Benign Prostatic Hyperplasia orprostatitis by administration of a homeopathically activated form ofantibodies to prostate specific antigen (PSA).

The S-100 protein is a cytoplasmic acidic calcium binding protein foundpredominantly in the gray matter of the brain, primarily in glia andSchwann cells. The protein exists in several homo- or heterodimericisoforms consisting of two immunologically distinct subunits, alpha andbeta. The S-100 protein has been suggested for use as an aid in thediagnosis and assessment of brain lesions and neurological damage due tobrain injury, as in stroke. Yardan et al., Usefulness of S100B Proteinin Neurological Disorders, J Pak Med Assoc Vol. 61, No. 3, March 2011,which is incorporated herein by reference.

Ultra low doses of antibodies to S-100 protein have been shown to haveanxiolytic, anti-asthenic, anti-aggressive, stress-protective,anti-hypoxic, anti-ischemic, neuroprotective and nootropic activity. SeeCastagne V. et al., Antibodies to S100 proteins have anxiolytic-likeactivity at ultra-low doses in the adult rat, J Pharm Pharmacol. 2008,60(3):309-16; Epstein O. I., Antibodies to calcium-binding S1008 proteinblock the conditioning of long-term sensitization in the terrestrialsnail, Pharmacol Biochem Behav., 2009, 94(1):37-42; Voronina T. A. etal., Chapter 8. Antibodies to S-100 protein in anxiety-depressivedisorders in experimental and clinical conditions. In “Animal models inbiological psychiatry”, Ed. Kalueff A. V. N—Y, “Nova Science Publishers,Inc.”, 2006, pp. 137-152, all of which are incorporated herein byreference.

Ultra low doses of antibodies to gamma interferon have been shown to beuseful in the treatment and prophylaxis of treating a disease of viralorigination. See U.S. Pat. No. 7,572,441, which is incorporated hereinby reference in its entirety.

SUMMARY

In one aspect, the invention provides a combination pharmaceuticalcomposition comprising a) an activated-potentiated form of an antibodyto gamma interferon, and b) an activated-potentiated form of an antibodyto S-100 protein.

In one variant, the present invention provides a combinationpharmaceutical composition comprising a) an activated-potentiated formof an antibody to gamma interferon, and b) an activated-potentiated formof an antibody to S-100 protein, wherein the antibody is to the entiregamma interferon or fragments thereof.

In one variant, the invention provides a combination pharmaceuticalcomposition comprising a) an activated-potentiated form of an antibodyto gamma interferon, and b) an activated-potentiated form of an antibodyto S-100 protein, wherein the antibody to the S-100 protein is anantibody to the entire S-100 protein or fragments thereof.

In one variant, the combination pharmaceutical composition of thisaspect of the invention includes activated-potentiated form of anantibody to gamma interferon is in the form of a mixture of (C12, C30,and C50) or (C12, C30 and C200) homeopathic dilutions impregnated onto asolid carrier. The activated-potentiated form of an antibody to S-100protein is in the form of mixture of (C12, C30, and C50) or (C12, C30and C200) homeopathic dilutions may be subsequently impregnated onto thesolid carrier.

In another variant, the combination pharmaceutical composition of thisaspect of the invention includes the activated-potentiated form of anantibody to S-100 protein is in the form of mixture of (C12, C30, andC50) or (C12, C30 and C200) homeopathic dilutions impregnated onto asolid carrier. The activated-potentiated form of an antibody to gammainterferon is in the form of mixture of (C12, C30, and C50) or (C12, C30and C200) homeopathic dilutions may be subsequently impregnated onto thesolid carrier.

Preferably, the activated-potentiated form of an antibody to gammainterferon is a monoclonal, polyclonal or natural antibody, morepreferably, a polyclonal antibody. In one variant of this aspect of theinvention, the activated-potentiated form of an antibody to gammainterferon is prepared by successive centesimal dilutions coupled withshaking of every dilution.

Preferably, the activated-potentiated form of an antibody to S-100protein is a monoclonal, polyclonal or natural antibody, morepreferably, a polyclonal antibody. In one variant of this aspect of theinvention, the activated-potentiated form of an antibody to S-100protein is prepared by successive centesimal dilutions coupled withshaking of every dilution. Vertical shaking is specificallycontemplated.

In another aspect, the invention provides a method of treating a patientsuffering from multiple sclerosis by administration of a) anactivated-potentiated form of an antibody to gamma interferon, and b) anactivated-potentiated form of an antibody to S-100 protein. Preferably,the activated-potentiated form of an antibody to gamma interferon andthe activated-potentiated form of an antibody to S-100 protein areadministered in the form of combined pharmaceutical composition.

In another aspect, the invention provides a method of significantlydelaying the onset of symptoms in a patient suffering from multiplesclerosis by administration of a combination pharmaceutical compositionwherein the composition comprises a) an activated-potentiated form of anantibody to gamma interferon, and b) an activated-potentiated form of anantibody to S-100 protein.

In another aspect, the present invention further provides a method ofreducing the frequency of appearance of relapse in a patient sufferingfrom multiple sclerosis by administration of a combinationpharmaceutical composition wherein the composition comprises a) anactivated-potentiated form of an antibody to gamma interferon, and b) anactivated-potentiated form of an antibody to S-100 protein.

In one variant of the invention, there is provided administration offrom one to two unit dosage forms of the activated-potentiated form ofan antibody to gamma interferon, and from one to two unit dosage formsof the activated-potentiated form of an antibody to S-100 protein eachof the dosage form being administered from once daily to four timesdaily. Preferably, the one to two unit dosage forms of each of theactivated-potentiated forms of antibodies is administered twice daily.

In a preferred variant of this aspect of the invention, there isprovided administration of from one to two unit dosage forms, of thecombination composition comprising a) the activated-potentiated form ofan antibody to gamma interferon, and b) the activated-potentiated formof an antibody to S-100 protein, each of the dosage form beingadministered from once daily to four times daily. Preferably, one to twounit dosage forms are administered twice daily.

In another variant of this aspect of the invention, which is preferred,the combination is administered in the form of one unit dosage formcomprising a) the activated-potentiated form of an antibody to gammainterferon, and b) the activated-potentiated form of an antibody toS-100 protein, preferably twice daily.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Illustrates the immune response in pathogenesis of multiplesclerosis

FIG. 2—Shows the mean day of clinical symptoms onset

FIG. 3—Shows the severity of disease (points)

FIG. 4—Shows the severity of disease at different stages (points)

FIG. 5—Shows the frequency of appearance of relapse

DETAILED DESCRIPTION

The invention is defined with reference to the appended claims. Withrespect to the claims, the glossary that follows provides the relevantdefinitions.

The term “antibody” as used herein shall mean an immunoglobulin thatspecifically binds to, and is thereby defined as complementary with, aparticular spatial and polar organization of another molecule.Antibodies as recited in the claims may include a completeimmunoglobulin or fragment thereof, may be natural, polyclonal ormonoclonal, and may include various classes and isotypes, such as IgA,IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof mayinclude Fab, Fv and F(ab′)₂, Fab′, and the like. The singular “antibody”includes plural “antibodies”.

The term “activated-potentiated form” or “potentiated form”respectively, with respect to antibodies recited herein is used todenote a product of homeopathic potentization of any initial solution ofantibodies. “Homeopathic potentization” denotes the use of methods ofhomeopathy to impart homeopathic potency to an initial solution ofrelevant substance. Although not so limited, ‘homeopathic potentization”may involve, for example, repeated consecutive dilutions combined withexternal treatment, particularly (mechanical) shaking. In other words,an initial solution of antibody is subjected to consecutive repeateddilution and multiple vertical shaking of each obtained solution inaccordance with homeopathic technology. The preferred concentration ofthe initial solution of antibody in the solvent, preferably water or awater-ethyl alcohol mixture, ranges from about 0.5 to about 5.0 mg/ml.The preferred procedure for preparing each component, i.e. antibodysolution, is the use of the mixture of three aqueous or aqueous-alcoholdilutions of the primary matrix solution (mother tincture) of antibodiesdiluted 100¹², 100³⁰ and 100²⁰⁰ times, respectively, which is equivalentto centesimal homeopathic dilutions (C12, C30, and C200) or the use ofthe mixture of three aqueous or aqueous-alcohol dilutions of the primarymatrix solution of antibodies diluted 100¹², 100³⁰ and 100⁵⁰ times,respectively, which is equivalent to centesimal homeopathic dilutions(C12, C30 and C50). Examples of homeopathic potentization are describedin U.S. Pat. Nos. 7,572,441 and 7,582,294, which are incorporated hereinby reference in their entirety and for the purpose stated. While theterm “activated-potentiated form” is used in the claims, the term“ultra-low doses” is used in the examples. The term “ultra-low doses”became a term of art in the field of art created by study and use ofhomeopathically diluted and potentized form of substance. The term“ultra-low dose” or “ultra-low doses” is meant as fully supportive andprimarily synonymous with the term ‘activated-potentiated” form used inthe claims.

In other words, an antibody is in the “activated-potentiated” or“potentiated” form when three factors are present. First, the“activated-potentiated” form of the antibody is a product of apreparation process well accepted in the homeopathic art. Second, the“activated-potentiated” form of antibody must have biological activitydetermined by methods well accepted in modern pharmacology. And third,the biological activity exhibited by the “activated potentiated” form ofthe antibody cannot be explained by the presence of the molecular formof the antibody in the final product of the homeopathic process.

For example, the activated potentiated form of antibodies may beprepared by subjecting an initial, isolated antibody in a molecular formto consecutive multiple dilutions coupled with an external impact, suchas mechanical shaking. The external treatment in the course ofconcentration reduction may also be accomplished, for example, byexposure to ultrasonic, electromagnetic, or other physical factors. V.Schwabe “Homeopathic medicines”, M., 1967, U.S. Pat. Nos. 7,229,648 and4,311,897, which are incorporated by reference in their entirety and forthe purpose stated, describe such processes that are well acceptedmethods of homeopathic potentiation in the homeopathic art. Thisprocedure gives rise to a uniform decrease in molecular concentration ofthe initial molecular form of the antibody. This procedure is repeateduntil the desired homeopathic potency is obtained. For the individualantibody, the required homeopathic potency can be determined bysubjecting the intermediate dilutions to biological testing in thedesired pharmacological model. Although not so limited, ‘homeopathicpotentization” may involve, for example, repeated consecutive dilutionscombined with external treatment, particularly vertical (mechanical)shaking. In other words, an initial solution of antibody is subjected toconsecutive repeated dilution and multiple vertical shaking of eachobtained solution in accordance with homeopathic technology. Thepreferred concentration of the initial solution of antibody in thesolvent, preferably, water or a water-ethyl alcohol mixture, ranges fromabout 0.5 to about 5.0 mg/ml. The preferred procedure for preparing eachcomponent, i.e. antibody solution, is the use of the mixture of threeaqueous or aqueous-alcohol dilutions of the primary matrix solution(mother tincture) of antibodies diluted 100¹², 100³⁰ and 100²⁰⁰ times,respectively, which is equivalent to centesimal homeopathic dilutionsC12, C30 and C200 or the mixture of three aqueous or aqueous-alcoholdilutions of the primary matrix solution (mother tincture) of antibodiesdiluted 100¹², 100³⁰ and 100⁵⁰ times, respectively, which is equivalentto centesimal homeopathic dilutions C12, C30 and C50. Examples of how toobtain the desired potency are also provided, for example, in U.S. Pat.Nos. 7,229,648 and 4,311,897, which are incorporated by reference forthe purpose stated. The procedure applicable to the “activatedpotentiated” form of the antibodies described herein is described inmore detail below.

There has been a considerable amount of controversy regardinghomeopathic treatment of human subjects. While the present inventionrelies on accepted homeopathic processes to obtain the“activated-potentiated” form of antibodies, it does not rely solely onhomeopathy in human subjects for evidence of activity. It has beensurprisingly discovered by the inventor of the present application andamply demonstrated in the accepted pharmacological models that thesolvent ultimately obtained from consecutive multiple dilution of astarting molecular form of an antibody has definitive activity unrelatedto the presence of the traces of the molecular form of the antibody inthe target dilution. The “activated-potentiated” form of the antibodyprovided herein are tested for biological activity in well acceptedpharmacological models of activity, either in appropriate in vitroexperiments, or in vivo in suitable animal models. The experimentsprovided further below provide evidence of biological activity in suchmodels. Human clinical studies also provide evidence that the activityobserved in the animal model is well translated to human therapy. Humanstudies have also provided evidence of availability of the “activatedpotentiated” forms described herein to treat specified human diseases ordisorders well accepted as pathological conditions in the medicalscience.

Also, the claimed “activated-potentiated” form of antibody encompassesonly solutions or solid preparations the biological activity of whichcannot be explained by the presence of the molecular form of theantibody remaining from the initial, starting solution. In other words,while it is contemplated that the “activated-potentiated” form of theantibody may contain traces of the initial molecular form of theantibody, one skilled in the art could not attribute the observedbiological activity in the accepted pharmacological models to theremaining molecular form of the antibody with any degree of plausibilitydue to the extremely low concentrations of the molecular form of theantibody remaining after the consecutive dilutions. While the inventionis not limited by any specific theory, the biological activity of the“activated-potentiated’ form of the antibodies of the present inventionis not attributable to the initial molecular form of the antibody.Preferred is the “activated-potentiated” form of antibody in liquid orsolid form in which the concentration of the initial molecular form ofthe antibody is below the limit of detection of the accepted analyticaltechniques, such as capillary electrophoresis and High PerformanceLiquid Chromatography. Particularly preferred is the“activated-potentiated” form of antibody in liquid or solid form inwhich the concentration of the initial molecular form of the antibody isbelow the Avogadro number. In the pharmacology of molecular forms oftherapeutic substances, it is common practice to create a dose-responsecurve in which the level of pharmacological response is plotted againstthe concentration of the active drug administered to the subject ortested in vitro. The minimal level of the drug which produces anydetectable response is known as a threshold dose. It is specificallycontemplated and preferred that the “activated-potentiated” form of theantibodies contains molecular antibody, if any, at a concentration belowthe threshold dose for the molecular form of the antibody in the givenbiological model.

Experimental animal models of multiple sclerosis are known. For example,Experimental autoimmune encephalomyelitis, sometimes ExperimentalAllergic Encephalomyelitis (EAE) is an animal model of inflammatorydemyelinating diseases of the central nervous system. It is mostly usedwith rodents and knockout mice. This experimental model is widelystudied as an animal model of the human central nervous systemdemyelinating diseases, such as multiple sclerosis. It is possible toinduce EAE in sensory animals by homogenate of homologous orheterologous cerebral tissue, purified myelin or proteins beingintroduced into the animal's composition (Raine, et al., Ann. NY Acad.Sci., 1984, 436: 33-51; McCombe, et al., J. Neuroimmunol. 1994; 51 (2):153-167).

At the present time, more than 20 myelin proteins have been describedwhich possess immunogenic properties and most frequently are used forinducting EAE (Baumann, et al., Physiol. Rev. 2001, 81(2): 871-927).Examples include: basic myelin protein (“MBP”) (Hashim, Immunol. Rev.,1978, 39:60-107); proteolipid protein (“PLP”) (Bradt, et al., BrainPathol., 1996, 6(3): 303-311; Tuohy, Neurochem. Res., 1994, 19(8):935-944); glycoproteins: myelin associated glycoprotein (“MAG”) (Weerthet al, 1999); myelin oligodendrocyte glycoprotein (“MOG”); andoligodendrocyte specific protein (“OSP”) (Stevens, et al., J. Immunol.,1999; 162 (12): 7501-7509). It is recognized that, not only wholeproteins, but also specific fragments of these proteins are capable ofcausing EAE when introduced into animals. The most commonly usedantigens in rodents are spinal cord homogenate (SCH), purified myelin,myelin protein such as MBP, PLP and MOG or peptides of these proteins,all resulting in distinct models with different disease characteristicsregarding both immunology and pathology.

The most adequate model MS is caused in EAE models by introducinghomogenate of spinal cord (Gilerovich et al, 2010; Zhabotinskiy, Joffe,1975; Zhitnukhin et al, 2008; Sinha et al, 2009). In this model, as withthe initial disease, immune response is made to all components ofmyelin: inflammation is induced with subsequent demyelination,degeneration of axons and then the nerve cells themselves (Gilerovich etal, 2010; Sinha et al, 2009). A pathologic chain of events is manifestedwith development of pareses, paralyzes and other disease symptoms.

In the development of EAE, it was possible to distinguish fourstages: 1. Sensitization of T-lymphocytes on the periphery under theeffect of cerebral antigen with CFA and increase in the permeability ofGEB. 2. Migration of activated T-cells to the CNS and activation ofantigen-representing cells (astrocytes and microglia) directly in thebrain. These two stages correspond to the latent (induction) period whenclinical manifestations are not observed. 3. Development of autoimmuneand inflammatory reactions in the brain, which leads to demyelination ofnerve fibers (clinical period). 4. Suppression of pathologic processesand repair of damaged tissues (recovery phase). During this period,neurologic and motor disorders are smoothed in the majority of animalsand partial or complete recovery ensues, after which animals areresistant to the repeated induction of EAE. The average duration of EAEis 30 days: latent stage, clinical stage and recovery stage. Theduration of each stage is usually 7-10 days.

Analogous phases of development of the pathologic process in the CNS arealso observed with multiple sclerosis. See FIG. 1—Immune response inpathogenesis of multiple sclerosis (Wiendl & Kieseier, 2003).

The present invention provides a combination pharmaceutical compositioncomprising a) an activated-potentiated form of an antibody to gammainterferon, and b) an activated-potentiated form of an antibody to S-100protein. As set forth herein above, each of the individual components ofthe combination is generally known for its own individual medical uses.However, the inventors of the present patent application surprisinglydiscovered that administration of the combination remarkably delays theonset of symptoms and reduces the probability of relapse in patientswith multiple sclerosis.

The combination pharmaceutical composition in accordance with thisaspect of the invention may be in the liquid form or in solid form. Eachof the activated potentiated forms of the antibodies included in thepharmaceutical composition is prepared from an initial molecular form ofthe antibody via a process accepted in homeopathic art. The startingantibodies may be monoclonal, or polyclonal antibodies prepared inaccordance with known processes, for example, as described inImmunotechniques, G. Frimel, M., “Meditsyna”, 1987, p. 9-33; “Hum.Antibodies. Monoclonal and recombinant antibodies, 30 years after” byLaffly E., Sodoyer R. —2005—Vol. 14. —N 1-2. P. 33-55, both incorporatedherein by reference.

Monoclonal antibodies may be obtained, e.g., by means of hybridomatechnology. The initial stage of the process includes immunization basedon the principles already developed in the course of polyclonal antiserapreparation. Further stages of work involve the production of hybridcells generating clones of antibodies with identical specificity. Theirseparate isolation is performed using the same methods as in the case ofpolyclonal antisera preparation.

Polyclonal antibodies may be obtained via active immunization ofanimals. For this purpose, for example, suitable animals (e.g. rabbits)receive a series of injections of the appropriate antigen, either S-100protein or gamma interferon. The animals' immune system generatescorresponding antibodies, which are collected from the animals in aknown manner. This procedure enables preparation of a monospecificantibody-rich serum.

If desired, the serum containing antibodies may be purified, for exampleby using affine chromatography, fractionation by salt precipitation, orion-exchange chromatography. The resulting purified, antibody-enrichedserum may be used as a starting material for the preparation of theactivated-potentiated form of the antibodies. The preferredconcentration of the resulting initial solution of antibody in thesolvent, preferably water or a water-ethyl alcohol mixture, ranges fromabout 0.5 to about 5.0 mg/ml.

The preferred procedure for preparing each component of the combinationdrug according to the present invention is the use of the mixture ofthree aqueous-alcohol dilutions of the primary matrix solution ofantibodies diluted 100¹², 100³⁰ and 100⁵⁰ times, respectively, which isequivalent to centesimal homeopathic dilutions C12, C30, and C50 ordiluted 100¹², 100³⁰ and 100²⁰⁰ times, respectively, which is equivalentto centesimal homeopathic dilutions C12, C30 and C200. To prepare asolid dosage form, a solid carrier is treated with the desired dilutionobtained via the homeopathic process. To obtain a solid unit dosage formof the combination of the invention, the carrier mass is impregnatedwith each of the dilutions. Both orders of impregnation are suitable toprepare the desired combination dosage form.

In a preferred embodiment, the starting material for the preparation ofthe activated potentiated form that comprise the combination of theinvention is polyclonal, animal-raised antibody to the correspondingantigen, namely, gamma interferon or S-100 protein. To obtain theactivated-potentiated form of polyclonal antibodies to gamma interferon,the desired antigen may be injected as immunogen into a laboratoryanimal, preferably, rabbits. Polyclonal antibodies to gamma interferonmay be obtained using the whole molecule of gamma interferon of thefollowing sequence:

SEQ. ID. NO. 1.Met Lys Tyr Thr Ser Tyr Ile Leu Ala Phe Gln Leu Cys Ile Val 1               5                   10                  15Leu Gly Ser Leu Gly Cys Tyr Cys Gln Asp Pro Tyr Val Lys Glu 16              20                  25                  30Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val 31              35                  40                  45Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys 46              50                  55                  60Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe 61              65                  70                  75Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln 76              80                  85                  90Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe 91              95                 100                 105Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn106             110                 115                 120Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu121             125                 130                 135Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly136             140                 145                 150Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Arg Ala Ser151             155                 160                 165 Gln 166Polyclonal antibodies to gamma interferon may be obtained using thewhole molecule of gamma interferon of the following sequence:

SEQ. ID. NO. 2Met Lys Tyr Thr Ser Tyr Ile Leu Ala Phe Gln Leu Cys Ile Val 1               5                   10                  15Leu Gly Ser Leu Gly Cys Tyr Cys Gln Asp Pro Tyr Val Lys Glu 16              20                  25                  30Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val 31              35                  40                  45Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys 46              50                  55                  60Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe 61              65                  70                  75Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln 76              80                  85                  90Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe 91              95                 100                 105Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn106             110                 115                 120Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu121             125                 130                 135Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly136             140                 145                 150Lys Arg Lys Arg Ser Gln Met Leu Phe Gln Gly Arg Arg Ala Ser151             155                 160                 165 Gln 166

The use of gamma interferon fragments as antigen is also contemplated.The suitable sequence for such antigen is as follow:

SEQ. ID. NO 3                        Ile Leu Ala Phe Gln Leu Cys Ile Val                         7           10                  15Leu Gly Ser Leu Gly Cys Tyr Cys Gln Asp Pro Tyr Val Lys Glu 16              20                  25                  30Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val 31              35                  40                  45Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile 46              50                  55 SEQ. ID. NO 4                                Gln Asp Pro Tyr Val Lys Glu                                24                       30Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val 31              35                  40                  45Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys 46              50                  55                  60Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe 61              65                  70                  75Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln 76              80                  85                  90Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe 91              95                 100                 105Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn106             110                 115                 120Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu121             125                 130                 135Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly136             140                 145                 150Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Arg Ala Ser151             155                 160                 165 Gln 166SEQ. ID. NO 5                                Gln Asp Pro Tyr Val Lys Glu                                24                       30Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val 31              35                  40                  45Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys 46              50                  55                  60Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe 61              65                  70                  75Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln 76              80                  85                  90Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe 91              95                 100                 105Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn106             110                 115                 120Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu121             125                 130                 135Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly136             140                 145                 150Lys Arg Lys Arg Ser Gln Met Leu Phe Gln Gly Arg Arg Ala Ser151             155                 160                 165 Gln 166SEQ. ID. NO 6                                Gln Ser Gln Ile Val Ser Phe                                69                      75Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln 76              80                  85                  90Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe 91              95                 100                 105Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn106             110                 115                 120 Tyr Ser Val121     123 SEQ. ID. NO 7                                    Met Asn Val Lys Phe Phe                                    100                 105Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn106             110                 115                 120Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu121             125                 130                 135Leu Ile Gln Val Met Ala Glu Leu Ser Pro136             140                 145 SEQ. ID. NO 8    Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe    92          95                  100                 105Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn106             110                 115                 120Tyr Ser Val Thr Asp Leu Asn Val Gln Arg121             125                 130 SEQ. ID. NO 9        Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu         123    125                 130                 135Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala136             140                 145     147 SEQ. ID. NO 10                Ser Tyr Ile Leu Ala Phe Gln Leu Cys Ile Val                  5                   10 15Leu Gly Ser Leu Gly Cys Tyr Cys Gln Asp Pro Tyr Val Lys Glu 16              20                  25                  30Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val 31              35                  40                  45SEQ. ID. NO 11            Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe             94                      100 105Asn Ser Asn Lys Lys Lys Arg Asp Asp 106             110             114

Polyclonal antibodies to gamma interferon may be obtained using themolecule of recombinant gamma interferon of one of the followingsequences:

SEQ. ID. NO 12                            Met Gln Asp Pro Tyr Val Lys Glu                                24                       30Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val 31              35                  40                  45Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys 46              50                  55                  60Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe 61              65                  70                  75Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln 76              80                  85                  90Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe 91              95                 100                 105Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn106             110                 115                 120Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu121             125                 130                 135Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly136             140                 145                 150Lys Arg Lys Arg Ser Gln Met Leu Phe Gln Gly Arg Arg Ala Ser151             155                 160                 165 Gln 166SEQ. ID. NO 13                            Met Gln Asp Pro Tyr Val Lys Glu                                24                       30Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val 31              35                  40                  45Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys 46              50                  55                  60Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe 61              65                  70                  75Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln 76              80                  85                  90Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe 91              95                 100                 105Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn106             110                 115                 120Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu121             125                 130                 135Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly136             140                 145                 150Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Arg Ala Ser151             155                 160                 165 Gln 166

The exemplary procedure for preparation of the starting polyclonalantibodies to human gamma interferon may be described as follows. In 7-9days before blood sampling, 1-3 intravenous injections of the desiredantigen are made to the rabbits to increase the level of polyclonalantibodies in the rabbit blood stream. Upon immunization, blood samplesare taken to test the antibody level. Typically, the maximum level ofimmune reaction of the soluble antigen is achieved within 40 to 60 daysafter the first injection of the antigen. Upon completion of the firstimmunization cycle, rabbits have a 30-day rehabilitation period, afterwhich re-immunization is performed with another 1-3 intravenousinjections.

To obtain antiserum containing the desired antibodies, the immunizedrabbits' blood is collected from rabbits and placed in a 50 mlcentrifuge tube. Product clots formed on the tube sides are removed witha wooden spatula, and a rod is placed into the clot in the tube center.The blood is then placed in a refrigerator for one night at thetemperature of about 40° C. On the following day, the clot on thespatula is removed, and the remaining liquid is centrifuged for 10 minat 13,000 rotations per minute. Supernatant fluid is the targetantiserum. The obtained antiserum is typically yellow. 20% of NaN₃(weight concentration) is added in the antiserum to a finalconcentration of 0.02% and stored before use in frozen state at thetemperature of −20° C. or without NaN₃ at the temperature of −70° C. Toseparate the target antibodies to gamma interferon from the antiserum,the following solid phase absorption sequence is suitable:

10 ml of the antiserum of rabbits is diluted twofold with 0.15 M NaCl,after which 6.26 g Na₂SO₄ is added, mixed and incubated for 12-16 hoursat 4° C. The sediment is removed by centrifugation, diluted in 10 ml ofphosphate buffer and dialyzed against the same buffer during one nightat ambient temperature. After the sediment is removed, the solution isapplied to a DEAE-cellulose column balanced by phosphate buffer. Theantibody fraction is determined by measuring the optical density of theeluate at 280 nm.

The isolated crude antibodies are purified using affine chromatographymethod by attaching the obtained antibodies to gamma interferon locatedon the insoluble matrix of the chromatography media, with subsequentelution by concentrated aqueous salt solutions.

The resulting buffer solution is used as the initial solution for thehomeopathic dilution process used to prepare the activated potentiatedform of the antibodies. The preferred concentration of the initialmatrix solution of the antigen-purified polyclonal rabbit antibodies togamma interferon is 0.5 to 5.0 mg/ml, preferably, 2.0 to 3.0 mg/ml.

The brain-specific S100 protein, expressed by neurons and glial cells(astrocytes and oligodendrocytes), directly or through interactions withother proteins executes in the CNS a number of functions directed atmaintaining normal brain functioning, including affecting learning andmemory processes, growth and viability of neurons, regulation ofmetabolic processes in neuronal tissues and others. To prepare theactivated-potentiated form of antibodies, an antiserum to brain-specificS-100 protein may be removed from the brain tissue of a bull andprocessed as follows:

-   -   the bull brain tissue frozen in liquid nitrogen is converted        into powder using a specialized mill;    -   proteins are extracted in the ratio of 1:3 (weight/volume) using        an extracting buffer with homogenization;    -   the homogenate is heated for 10 min at 60° C. and then cooled to        4° C. in an ice bath;    -   thermolabile proteins are removed by centrifugation;    -   ammonium sulfate fractionation is carried out in stages, with        subsequent removal of precipitated proteins;    -   the fraction containing S-100 protein is precipitated using 100%        saturated ammonium sulfate accomplished by pH drop to 4.0; the        desired fraction is collected by centrifugation;    -   the precipitate is dissolved in a minimum buffer volume        containing EDTA and mercaptoethanol, the precipitate is dialyzed        with deionized water and lyophilized;    -   fractionation of acidic proteins is followed by chromatography        in ion-exchanging media, DEAE-cellulose DE-52 and then        DEAE-sephadex A-50;    -   the collected and dialyzed fractions, which contain S-100        protein, are divided according to molecular weight by gel        filtration on sephadex G-100;    -   purified S-100 protein is dialyzed and lyophilized.        The molecular weight of the purified brain-specific S-100        protein is 21000 D.        The polyclonal antibodies to S-100 protein may also be obtained        by a similar methodology to the methodology described for gamma        interferon antibodies using an adjuvant. The entire molecule of        S-100 protein may be used as immunogen (antigen) for rabbits'        immunization.

Bovine S100B (SEQ. ID. NO. 14)Met Ser Glu Leu Glu Lys Ala Val Val Ala Leu Ile Asp Val Phe 1               5                   10                  15His Gln Tyr Ser Gly Arg Glu Gly Asp Lys His Lys Leu Lys Lys 16              20                  25                  30Ser Glu Leu Lys Glu Leu Ile Asn Asn Glu Leu Ser His Phe Leu 31              35                  40                  45Glu Glu Ile Lys Glu Gln Glu Val Val Asp Lys Val Met Glu Thr 46              50                  55                  60Leu Asp Ser Asp Gly Asp Gly Glu Cys Asp Phe Gln Glu Phe Met 61              65                  70                  75Ala Phe Val Ala Met Ile Thr Thr Ala Cys His Glu Phe Phe Glu 76              80                  85                  90 His Glu 91  92 Human S100B (SEQ. ID. 15)Met Ser Glu Leu Glu Lys Ala Met Val Ala Leu Ile Asp Val Phe 1               5                   10                  15His Gln Tyr Ser Gly Arg Glu Gly Asp Lys His Lys Leu Lys Lys 16              20                  25                  30Ser Glu Leu Lys Glu Leu Ile Asn Asn Glu Leu Ser His Phe Leu 31              35                  40                  45Glu Glu Ile Lys Glu Gln Glu Val Val Asp Lys Val Met Glu Thr 46              50                  55                  60Leu Asp Asn Asp Gly Asp Gly Glu Cys Asp Phe Gln Glu Phe Met 61              65                  70                  75Ala Phe Val Ala Met Val Thr Thr Ala Cys His Glu Phe Phe Glu 76              80                  85                  90 His Glu 91  92 Human S100A1 (SEQ. ID. No. 16)Met Gly Ser Glu Leu Glu Thr Ala Met Glu Thr Leu Ile Asn Val 1               5                   10                  15Phe His Ala His Ser Gly Lys Glu Gly Asp Lys Tyr Lys Leu Ser 16              20                  25                  30Lys Lys Glu Leu Lys Glu Leu Leu Gln Thr Glu Leu Ser Gly Phe 31              35                  40                  45Leu Asp Ala Gln Lys Asp Val Asp Ala Val Asp Lys Val Met Lys 46              50                  55                  60Glu Leu Asp Glu Asn Gly Asp Gly Glu Val Asp Phe Gln Glu Tyr 61              65                  70                  75Val Val Leu Val Ala Ala Leu Thr Val Ala Cys Asn Asn Phe Phe 76              80                  85                  90Trp Glu Asn Ser  91          94 Bovine S100A1 (SEQ. ID. NO. 17)Met Gly Ser Glu Leu Glu Thr Ala Met Glu Thr Leu Ile Asn Val 1               5                   10                  15Phe His Ala His Ser Gly Lys Glu Gly Asp Lys Tyr Lys Leu Ser 16              20                  25                  30Lys Lys Glu Leu Lys Glu Leu Leu Gln Thr Glu Leu Ser Gly Phe 31              35                  40                  45Leu Asp Ala Gln Lys Asp Ala Asp Ala Val Asp Lys Val Met Lys 46              50                  55                  60Glu Leu Asp Glu Asn Gly Asp Gly Glu Val Asp Phe Gln Glu Tyr 61              65                  70                  75Val Val Leu Val Ala Ala Leu Thr Val Ala Cys Asn Asn Phe Phe 76              80                  85                  90Trp Glu Asn Ser  91          94

To obtain brain-specific antiserum to separated brain-specific S-100protein, a mixture of purified S-100 protein (antigen) may be preparedin complex by methylated bull serum albumin as the medium with completeFreund's adjuvant, which is subcutaneously injected in the laboratoryanimal, rabbit, in the area of the spine in the quantity of 1-2 ml. Theantiserum may have a titer of 1:500-1:1000.

The activated potentiated form of each component of the combination maybe prepared from an initial solution by homeopathic potentization,preferably using the method of proportional concentration decrease byserial dilution of 1 part of each preceding solution (beginning with theinitial solution) in 9 parts (for decimal dilution), or in 99 parts (forcentesimal dilution), or in 999 parts (for millesimal dilution) of aneutral solvent, starting with a concentration of the initial solutionof antibody in the solvent, preferably, water or a water-ethyl alcoholmixture, in the range from about 0.5 to about 5.0 mg/ml, coupled withexternal impact. Preferably, the external impact involves multiplevertical shaking (dynamization) of each dilution. Preferably, separatecontainers are used for each subsequent dilution up to the requiredpotency level, or the dilution factor. This method is well-accepted inthe homeopathic art. See, e.g. V. Schwabe “Homeopathic medicines”, M.,1967, p. 14-29, incorporated herein by reference for the purpose stated.

For example, to prepare a 12-centesimal dilution (denoted C12), one partof the initial matrix solution of antibodies to gamma interferon withthe concentration of 3.0 mg/ml is diluted in 99 parts of neutral aqueousor aqueous-alcohol solvent (preferably, 15%-ethyl alcohol) and thenvertically shaked many times (10 and more) to create the 1st centesimaldilution (denoted as C1). The 2nd centesimal dilution (C2) is preparedfrom the 1st centesimal dilution C1. This procedure is repeated 11 timesto prepare the 12th centesimal dilution C12. Thus, the 12th centesimaldilution C12 represents a solution obtained by 12 serial dilutions ofone part of the initial matrix solution of antibodies to gammainterferon with the concentration of 3.0 mg/ml in 99 parts of a neutralsolvent in different containers, which is equivalent to the centesimalhomeopathic dilution C12. Similar procedures with the relevant dilutionfactor are performed to obtain dilutions C30, C50 and C200. Theintermediate dilutions may be tested in a desired biological model tocheck activity. The preferred activated potentiated forms for bothantibodies comprising the combination of the invention are a mixture ofC12, C30, and C50 dilutions or C12, C30 and C200 dilutions. When usingthe mixture of various homeopathic dilutions (primarily centesimal) ofthe active substance as biologically active liquid component, eachcomponent of the composition (e.g., C12, C30, C50, C200) is preparedseparately according to the above-described procedure until thenext-to-last dilution is obtained (e.g., until C11, C29, and C199respectively), and then one part of each component is added in onecontainer according to the mixture composition and mixed with therequired quantity of the solvent (e.g. with 97 parts for centesimaldilution).

It is possible to use the active substance as mixture of varioushomeopathic dilutions, e.g. decimal and/or centesimal (D20, C30, C100 orC12, C30, C50 or C12, C30, C200, etc.), the efficiency of which isdetermined experimentally by testing the dilution in a suitablebiological model, for example, in models described in the examplesherein.

In the course of potentiation and concentration decrease, the verticalshaking may be substituted for external exposure to ultrasound,electromagnetic field or any similar external impact procedure acceptedin the homeopathic art.

Preferably, the pharmaceutical composition of the invention may be inthe form of a liquid or in the solid unit dosage form. The preferredliquid form of the pharmaceutical composition is a mixture, preferably,at a 1:1 ratio of the activated potentiated form of antibodies to gammainterferon and the activated potentiated form of antibodies to S-100protein. The preferred liquid carrier is water or water-ethyl alcoholmixture.

The solid unit dosage form of the pharmaceutical composition of theinvention may be prepared by impregnating a solid, pharmaceuticallyacceptable carrier with the mixture of the activated potentiated formaqueous or aqueous-alcohol solutions of active components that aremixed, preferably in 1:1 ratio. Alternatively, the carrier may beimpregnated consecutively with each requisite dilution. Both orders ofimpregnation are acceptable.

Preferably, the pharmaceutical composition in the solid unit dosage formis prepared from granules of the pharmaceutically acceptable carrierwhich was previously saturated with the aqueous or aqueous-alcoholicdilutions of the activated potentiated form of antibodies to gammainterferon and the activated potentiated form of antibodies to S-100protein. The solid dosage form may be in any form known in thepharmaceutical art, including a tablet, a capsule, a lozenge, andothers. As an inactive pharmaceutical ingredients one can use glucose,sucrose, maltose, amylum, isomaltose, isomalt and other mono-olygo- andpolysaccharides used in manufacturing of pharmaceuticals as well astechnological mixtures of the above mentioned inactive pharmaceuticalingredients with other pharmaceutically acceptable excipients, forexample isomalt, crospovidone, sodium cyclamate, sodium saccharine,anhydrous citric acid etc), including lubricants, disintegrants, bindersand coloring agents. The preferred carriers are lactose and isomalt. Thepharmaceutical dosage form may further include standard pharmaceuticalexcipients, for example, microcrystalline cellulose and magnesiumstearate.

To prepare the solid oral form, 100-300 μm granules of lactose areimpregnated with aqueous or aqueous-alcoholic solutions of the activatedpotentiated form of antibodies to histamine, activated-potentiated formof antibodies to gamma-interferon and the activated potentiated form ofantibodies to S-100 protein in the ratio of 1 kg of antibody solution to5 or 10 kg of lactose (1:5 to 1:10). To effect impregnation, the lactosegranules are exposed to saturation irrigation in the fluidized boilingbed in a boiling bed plant (e.g. “Hüttlin Pilotlab” by Hüttlin GmbH)with subsequent drying via heated air flow at a temperature below 40° C.The estimated quantity of the dried granules (10 to 34 weight parts)saturated with the activated potentiated form of antibodies is placed inthe mixer, and mixed with 25 to 45 weight parts of “non-saturated” purelactose (used for the purposes of cost reduction and simplification andacceleration of the technological process without decreasing thetreatment efficiency), together with 0.1 to 1 weight parts of magnesiumstearate, and 3 to 10 weight parts of microcrystalline cellulose. Theobtained tablet mass is uniformly mixed, and tableted by direct drypressing (e.g., in a Korsch-XL 400 tablet press) to form 150 to 500 mground pills, preferably, 300 mg. After tableting, 300 mg pills areobtained that are saturated with aqueous-alcohol solution (3.0-6.0mg/pill) of the combination of the activated potentiated form ofantibodies to gamma interferon and the activated potentiated form ofantibodies to S-100 protein. Each component of the combination used toimpregnate the carrier is in the form of a mixture of centesimalhomeopathic dilutions C12, C30, and C50 or a mixture of centesimalhomeopathic dilutions C12, C30 and C200.

While the invention is not limited to any specific theory, it isbelieved that the activated potentiated form of the antibodies describedherein do not contain the molecular form of the antibody in an amountsufficient to have biological activity attributed to such molecularform. The biological activity of the combination drug (combinationpharmaceutical composition) of the invention is amply demonstrated inthe appended examples.

The present invention further provides a method of significantlydelaying the onset of symptoms of multiple sclerosis, said methodcomprising administering a combination pharmaceutical compositioncomprising a) an activated-potentiated form of an antibody to gammainterferon, and b) an activated-potentiated form of an antibody to S-100protein.

The present invention further provides a method of reducing theprobability of relapse of episodes in a patient suffering from multiplesclerosis by administration of a combination pharmaceutical compositioncomprising a) an activated-potentiated form of an antibody to gammainterferon, and b) an activated-potentiated form of an antibody to S-100protein.

Preferably, for the purpose of treatment, the combination of theinvention is administered from once daily to four times daily,preferably twice daily, each administration including one or twocombination unit dosage forms.

The invention is further illustrated with reference to the appendednon-limiting examples.

EXAMPLES Example 1

In this study female rats of the Wistar line (200-220 g) were used. EAEwas induced in them by single inoculation of encephalitogenic mixturebased on 100 mH of homogenate of homologous spinal cord, 0.2 ml CFA(content of killed mycobacteria 5 mg/ml) and 0.2 ml of physiologicalsolution to one animal. The EGS was administered subcutaneously (at thebase of the tail along the tail vein) under light ether anesthesia inthe amount of 0.4 ml (0.2 ml on the right and left) (Abdurasulova, I.N., et al, 2004; Zhitnukhin, Y. L., et al, 2008; Serebryanaya, N. B., etal, 2010). The following were administered intragastrically 2 times aday at 7-hour intervals over 30 days, beginning from the day of EAEinduction: (a) Ultra low dose of antibodies to S-100 protein(hereinafter “ULD Abs to S-100 protein”) (n=10, 2.5 ml/kg/day); (b)ultra low dose of antibodies to gamma interferon (hereinafter “ULD Absto γ-IFN”) (n=10, 2.5 ml/kg/day); (c) the combination of ULD Abs toS-100 protein and ULD Abs to γ-IFN (hereinafter “the complexcombination”) (n=10, 5 ml/kg/day) and (d) the distilled water (control;n=10, 5 ml/kg/day). As a reference preparation, the immunomodulatorCopaxone® (Teva, Israel) was used, which was administeredintramuscularly at the dose of 4 mg/kg, from the 2nd to the 25th dayafter EAE induction. The results are shown on FIG. 2-5.

Clinical symptoms of experimental autoimmune encephalomyelitis (EAE)were assessed daily for 30 days starting on the day of induction ofexperimental autoimmune encephalomyelitis (EAE). Examination of each ratwas carried out immediately before administration of drugs under study.In case of rapid disease progression the clinical symptoms were assessedtwice daily.

The severity of neurological abnormalities was assessed in points:presence of muscle weakness, tremor (0.5 point); resistant paresis (1point); paralysis (1.5 points). Clinical Index (CI) was calculated as asum of the symptoms for 4 limbs. In addition, CI was designated as zeroif visible clinical signs of neurologic abnormalities were absent, anddesignated as 6 in case of animal death. The maximal CI value was takeninto account at all days. Cumulative index for each rat calculated as asum of individual CI over total disease period (30 days) was recorded.The 30-day disease period was divided into the following phases: latentphase (1-10 days); clinical manifestation phase (11-18 days); andrecovery phase (19-30 days).

To assess the efficacy of drugs under study in the model of Experimentalautoimmune encephalomyelitis (EAE), the following parameters wereassessed in each study group: 1) time of disease onset (days); 2)changes of mean severity of the disease over time (CI, points); 3) meanseverity of the disease at different phases (CI, points) 4) total numberof relapses, return of disease symptoms (%).

ULD Abs to S-100 protein moved back the onset of manifestation ofclinical symptoms of disease periods, both in comparison with thenegative control (p<0.05) and in comparison with Copaxone (p>0.05). Theonset of disease in two groups that received ULD Abs to γ-IFN and in agroup that received Copaxone did not differ from the control. See FIG.2. At the same time, in animals that were administered the complexcombination, the tendency was observed to shortening of period ofappearance of clinical signs of multiple sclerosis in comparison withthe control. It should be noted that statistically significantdifferences were revealed in the time of disease onset period betweenthe group receiving ULD Abs to S-100 protein and the group receiving thecomplex combination.

The proportion of animals with severe course of disease (>3 points) wasalso lower in the group receiving ULD Abs to S-100 protein. In thegroups that received the complex combination the percentage of animalswith severe course was higher than in the other groups studied in alldisease periods. See FIG. 3.

In connection with the fact that, at various phases in the developmentof multiple sclerosis, different mechanisms of EAE pathogenesis wereactivated, the effect of the preparations on disease severity in thelatent period, in the development of clinical signs phase and in therecovery phase was analyzed. Administration of ULD Abs to S-100 proteinsignificantly contributed to reducing the average points ofmanifestation of clinical signs of disease in the clinical manifestationstage and in the recovery stage, while Copaxone and ULD Abs to γ-IFNsignificantly reduced the severity of the clinical symptoms only in thelast phase. The complex combination, in comparison with the control andthe groups receiving ULD Abs to γ-IFN alone or ULD Abs to S-100 proteinalone, statistically significant increased the average points of theaffection of animals in the appearance of clinical signs of diseasestage and in the recovery stage. See FIG. 4.

It should be noted that in a portion of the animals, after completedisappearance of the clinical signs of disease, a certain similarity torelapse, was noted, that is, the return of clinical manifestations ofEAE. In the ULD Abs to S-100 protein group, in spite of its beneficialeffect on the clinical course of EAE (later period of disease onset,lesser severity of manifestation of clinical signs), the greatest numberof animals (25%) with relapses is noted. At the same time, in thecomplex combination group, which was characterized by early onset ofdisease and greater manifestation of pathologic process, no animalshowed the development of relapses. See FIG. 5.

Conclusions:

In the model of experimental autoimmune encephalomyelitis (EAE), ULD Absto S-100 protein had a beneficial effect on the course of the disease.ULD Abs to S-100 protein diminished the clinical signs of the diseaseduring an episode and reduced severity of symptoms. ULD Abs to γ-IFN andCopaxone comparison preparation did not have an effect on the course ofdecease in the chosen experimental model of multiple sclerosis, that isfor these substances, the magnitude of clinical signs of disease periodsand the severity of symptoms did not differ from the control (distilledwater).

Manifestation of the effects of preparations depended on the stage ofdevelopment of EAE (latent phase, clinical signs phase, recovery phase).

The effect of the complex combination differed from the effect of thecomponents, namely, from ULD Abs to S-100 protein or ULD Abs to γ-IFNalone. Use of the complex combination led to strengthening of the immuneresponse. The complex combination led to earlier onset of disease,increase in the portion of sick animals and severity of disease.

25% of animals in the ULD Abs to S-100 protein group and 15% of animalsin the ULD Abs to γ-IFN group showed relapse during the study period (30days). At the same time, relapse was not recorded in any animal in thecomplex combination group.

The most common course of multiple sclerosis is the relapsing-remittingsubtype, which is characterized by unpredictable attacks (relapses),followed by periods of relative remission with no new signs of diseaseactivity.

The primary aims of multiple sclerosis therapy are reducing the neuronaldamage during an acute attack and returning function after an attack, aswell as preventing new attacks leading to disability.

ULD Abs to S100 protein alone was able to ameliorate clinical symptomsof EAE during all phases of experimental disease that makes itpotentially useful in the treatment of multiple sclerosis attacks. Thecombination of ULD Abs to S100 protein+ULD Abs to γ-IFN completelyprevented return of clinical symptoms of EAE, and thus, can be used inthe treatment of multiples sclerosis in humans as a means of preventionof relapse. Thus, the combination of ULD Abs to S-100 protein+ULD Abs toγ-IFN is a promising preparation for treating multiple sclerosis, which,it is believed, by strengthening of immune response at the peak ofdisease development, can contribute to more effective rehabilitation andprevention of relapses.

Example 2

Sigma-1 receptor—an intracellular receptor localized in the cells ofcentral nervous system, the cells of the most of peripheral tissues, andimmune cells. It is believed that this receptor, through control ofhomeostasis of intracellular calcium, regulates intracellular signalingevents leading to activation of the corresponding transcription factorsand transcription of a whole gene family. Sigma-1 receptor is involvedin the pathogenesis of various diseases including, lemic andneurodegenerative conditions. In this regard, drugs influencing thisreceptor and the efficiency of interaction of ligands with this receptormay be regarded as effective drugs for the treatment of neuroinfectiousand neurodegenerative diseases.

The effect of the complex combination whose composition includesultra-low doses of S100 protein antibodies (mixture of homeopathicdilutions C12+C30+C50) and ultra-low doses of interferon gammaantibodies (mixture of homeopathic dilutions C12+C30+C50) in the ratio1:1, and also components of the composition (ultra-low doses of S100protein antibodies (mixture of homeopathic dilutions C12+C30+C50) (ULDAbs to S-100 protein) and ultra-low doses of interferon gamma antibodies(mixture of homeopathic dilutions C12+C30+C50) (ULD Abs to γ-IFN)), wereinvestigated in vitro on binding of standard ligand [³H]pentazocine withrecombinant human sigma 1 receptor and evaluated by radioligand method.As control, potentiated distilled water (mixture of homeopathicdilutions C12+C30+C50) (potentiated water) was tested.

20 μl of the complex combination or 10 μl of ULD Abs to S100 protein orULD Abs to γ-IFN was introduced in the incubation medium. Thus, thequantity of ULD Abs to S100 protein and ULD Abs to γ-IFN introduced intothe experimental well during testing of the complex combination wasidentical to the quantity of ULD Abs to S100 protein and ULD Abs toγ-IFN tested as monopreparations, thus making it possible to compare theeffectiveness of the complex combination with the separate componentsthat constitute the complex composition. Potentiated water wasintroduced in the incubation medium in the amount of 20 μl and 10 μl.

Then 160 μl (−200 μg of protein) of homogenate of Jurkat line cellmembranes (line of human leukemic T-lymphocytes) was introduced,followed by 20 μl of the tritium-labeled radioligand [³H]pentazocine (15nM).

For measuring nonspecific bonding, instead of the preparations orpotentiated water, 20 μl of the unlabeled ligand Haloperidol (10 μM) wasintroduced in the incubation medium.

Radioactivity was measured on a scintillation counter (Topcount,Packard) with use of a scintillation mixture (Microscint 0, Packard)after incubation for 120 minutes at 22° C. in 50 mM Tris-HCL buffer(pH=7.4) and filtration on fiberglass filters (GF/B, Packard). Specificbinding (in the experiment or control) was calculated as the differencebetween covalent (in experiment or control) and nonspecific bonding.

Results (in measurement of covalent binding) are represented as apercentage of specific binding inhibition in the control (potentiatedwater was used as the control) (see Table 4).

TABLE 4 Effect of test preparations and potentiated water on binding ofstandard radioligand [³H]pentazocine with recombinant human sigma 1receptor Quantity % of specific binding % of inhibition introduced in ofradioligand in control of radioligand experimental 1st 2nd Averagebinding in Experimental group well measurement measurement value controlComplex 20 μl 44.2 46.2 45.2 54.8 Combination ULD Abs to S-100 10 μl70.9 62.9 66.9 33.1 protein ULD Abs to γ-IFN 10 μl 158.9 149.8 154.3−54.3 Potentiated water 20 μl 98.1 75.8 86.9 13.1 Potentiated water 10μl 140.1 106.2 123.2 −23.2 % of specific bonding in control = (specificbonding in experiment/specific bonding in control) * 100%; % of specificbinding inhibition in control = 100% − (specific bonding inexperiment/specific bonding in control) * 100%).

Inhibition of over 50% shows that there exists a significant effect onbinding. Inhibition from 25% to 50% shows weak to moderate effects.Inhibition of less than 25% are considered insignificant in terms ofeffect on binding, and are within background limits.

Conclusion:

The complex combination more effectively inhibits binding of standardradioligand [³H]pentazocine with recombinant human sigma 1 receptor thanits separate components (ULD Abs to S100 protein or ULD Abs to γ-IFN).

ULD Abs to S100 protein, introduced in an experimental well in theamount of 10 μl, inhibits the binding of standard radioligand[³H]pentazocine with recombinant human sigma 1 receptor, but expressionof the effect is inferior to expression of the effect with the complexcombination.

ULD Abs to γ-IFN, introduced in an experimental well in the amount of 10μl, did not have an effect on the binding of standard radioligand[³H]pentazocine with recombinant human sigma 1 receptor.

Potentiated water, introduced in an experimental well in the amount of10 μl or 20 μl, did not have an effect on the binding of standardradioligand [³H]pentazocine with recombinant human sigma 1 receptor.

1-16. (canceled)
 17. A method of treating neurodegenerative disease,said method comprising administering to a patient in need thereof,substantially at the same time a) an activated-potentiated form of anantibody to gamma interferon and b) an activated-potentiated form of anantibody to S-100 protein.
 18. The method of claim 17, wherein saidneurodegenerative disease is multiple sclerosis.
 19. The method of claim17 or 18, wherein said activated-potentiated form of an antibody togamma interferon and said activated-potentiated form of an antibody toS-100 protein are administered in the form of a combined pharmaceuticalcomposition.
 20. A method of reducing the frequency of appearance ofrelapse in a patient suffering from multiple sclerosis by administrationof the combination pharmaceutical composition of claim
 1. 21. The methodof claim 19, wherein the combination pharmaceutical composition isadministered in one to two unit dosage forms, each of the dosage formbeing administered from once daily to four times daily.
 22. The methodof claims 19, wherein the combination pharmaceutical composition isadministered in one to two unit dosage forms, each of the dosage formbeing administered twice daily.
 23. A method of mitigating of multiplesclerosis attack, said method comprising administering to a patient inneed thereof an activated-potentiated form of an antibody to S-100protein. 24-26. (canceled)